Project 2 - Summary / Abstract An extended form of Ang I, [Angiotensin-(1-12), Ang-(1-12)] has been shown to generate Ang II through a non-renin pathway in rodents and humans. We have shown that chymase in humans accounts for the direct hydrolysis of Ang-(1-12) into Ang II and substantial co-expression of Ang-(1-12) and chymase in atrial myocytes in patients undergoing the MAZE procedure for control of atrial fibrillation (AF). This suggests that Ang-(1-12) may be the substrate from which chymase forms Ang II within atrial myocytes. In addition to formation of Ang II, chymase activates matrix metalloproteinases (MMPs). MMPs contribute to myofibrillar breakdown and remodeling of connexins (Cx), which are gap-junctions proteins important in cell-cell communication and electrical stability. Here we will explore expression versus entry of chymase in the cardiomyocyte during ischemia/reperfusion (I/R) and determine how chymase mediates Ang-(1-12) conversion to Ang II and MMP activation in the myocyte. Protection of the heart during I/R is an unmet need during cardiac surgery. Furthermore, an intracellular chymase-mediated mechanism can explain the relative failure of ACE inhibitors and Ang II type I receptor (AT1R) blockers as anti-arrhythmic agents and in myocardial protection, since these drugs will not affect intracellular chymase-mediated MMP activation and Ang II formation. Project 2 will characterize: 1) direct chymase expression in human myocytes with laser capture dissection and in isolated myocytes during hypoxia reoxygenation; 2) mast cell chymase entry into the cardiomyocyte; 3) chymase-mediated intracellular activation of MMPs and breakdown of connexins and myofibrils; and 4) chymase-mediated conversion of Ang I precursors [BAng25 and Ang-(1-12)] to Ang II. Studies in isolated myocytes in vitro will show how chymase causes breakdown of Cx (40/43) and myofibrils in myocytes. In humans, we show chymase inside atrial and LV myocytes and a marked increase in pericardial fluid chymase activity, which is a direct reflection of the cardiac renin-angiotensin system (RAS)/ chymase axis. We will relate pericardial fluid chymase/MMP profile to postoperative AF, and atrial and LV dysfunction. The incidence of AF and decrease in LV ejection fraction is very common in patients after mitral valve surgery for mitral regurgitation (MR). Project 2 will test the hypothesis that intracellular chymase-mediated MMP activation and - Ang II formation via Ang-(1-12) relates to increasing left atrial and LV dysfunction and occurrence of AF in patients post-surgery for isolated MR. To gain further insight into these mechanisms in the human (Aim 1), Aims 2 and 3 will study effects of hypoxia on HL-1 atrial cells in vitro.